Interferon-induced human (2&#39;-5&#39;) oligo a synthetase

ABSTRACT

Human DNA encoding enzymes having (2&#39;-5&#39;) oligo A synthetase has been sequenced. The amino acid sequences of the enzymes have been deduced. Antigenic peptides have been prepared and have been used to raise antibodies which recognize and immunoprecipitate the 40 kd, 46 kd, 67 kd and 100 kd forms of (2&#39;-5&#39;) oligo A synthetase. Methods of monitoring interferon activity in a subject are presented.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part of U.S. Ser. No. 833,212,filed Feb. 25, 1986, which is a continuation-in-part of U.S. Ser. No.601,782, filed Apr. 18, 1984, now abandoned the contents of both ofwhich are hereby incorporated by reference.

Throughout this application, various publications are referenced by thename of the author and date of publication within parentheses. Fullcitations for these references may be found at the end of thespecification listed in alphabetical order immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art as known to those skilled therein asof the date of the invention described and claimed herein.

Many of the biological effects of interferon (IFN) appear to be mediatedby the induction of new mRNAs and proteins in cells exposed to IFNs (forreview: Revel, 1984; Lebleu and Content, 1982; Baglioni and Nilsen,1983). Among these IFN-induced proteins, two groups appear particularlyimportant: 1) translation regulatory enzymes (ds RNA dependent proteinkinase and (2'-5') oligo A synthetase, (2'-5') oligo A-activatednuclease, 2-phosphodiesterase); and 2) cell surface antigens (HLA-A, B,C, B2-microglobulin, HLA-DR). Other cellular and excreted proteinsprobably play important roles as well (Weil et al., 1983; Chebath etal., 1983; Wallach et al., 1983). With the exception of the HLA genes(Malissen et al., 1982; Schamboeck et al., 1983), the structure andsometimes the function of the IFN-induced proteins is unknown and so isthe mechanism by which IFNs activate specifically these genes. Toaddress these questions, several cDNAs from IFN-induced genes have beenrecently cloned (Chebath et al., 1983; Merlin et al., 1983; Friedman etal., 1984; Samanta et al., 1984). We have, in particular, studied thecDNA and gene coding for the human (2'-5') oligo A synthetase (OASE), ads RNA-activated enzyme that converts ATP into ppp(A2'pA)n oligomers(Kerr and Brown, 1978) which in turn bind to and activate the latentRNAse F (Schmidt et al., 1978). The (2'-5') oligo A synthetase isstrongly induced in cells by all three types of human IFNs, and itsincrease is a good marker of IFN activity (Wallach et al., 1982). Theenzyme is induced during differentiation of hematopoietic cells, anddenotes an autocrine secretion of IFN-beta (Yarden et al., 1984). Theenzyme is similarly induced late in the S phase of synchronized embryofibroblasts (Wells and Mallucci, 1985). The enzyme activity drops whencell growth starts (Etienne-Smekens et al., 1983; Creasey et al., 1983)and appears to be involved in the antigrowth effect of IFN (Kimchi etal., 1981). Deficiency in the (2'-5') oligo A synthetase or in the(2'-5') oligo A-activated RNAse F has also been correlated with partialloss of the antiviral effects of IFNs (Salzberg et al., 1983; Epstein etal., 1981), although this is probably not the only mechanism by whichIFN inhibits virus growth (Lebleu and Content, 1982). The (2'-5') oligoA nucleotides have been detected in many eucaryotic cells and even inbacteria (Laurence et al., 1984) and the synthetase is likely to be awide-spread enzyme. The enzyme has been purified from mouse (Doughertyet al., 1980) and human cells (Yand et al., 1981); Revel et al., 1981);a large and a small form of the enzyme have been observed (Revel et al.,1982; St. Laurent et al., 1983) but their structures were notelucidated.

The (2'-5') oligo A synthetase, induced in cells exposed to IFNs(Hovanessian et al., 1977; Zilberstein et al., 1978) has a number ofunusual properties. Its main activity is the synthesis from ATP of5'triphosphorylated short oligo A chains (of up to 15 A, with mainlydimers to pentamers), but in contrast to other RNA polymerases, it addsadenylate or one other nucleotide specifically to the 2'OH of adenylatein oligo A (Kerr and Brown, 1978; Samanta et al., 1980), or to other(oligo) nucleotides with a free 2'OH adenylate such as NAD Ball, 1980)or even tRNA (Ferbus et al., 1981). To be active, the enzyme has to bindto double-stranded RNA stretches of minimum 50 bp (Minks et al., 1979),and must therefore possess several binding sites: for nucleotidetriphosphates, for 2'OH adenosine polynucleotides and for doublestranded RNA. The enzyme binds to 2', 5' ADP-Sepharose (Johnston et al.,1980), to poly (rI)(rC)-Agarose (Hovanessian et al., 1977) and toCibacron Blue-Sepharose (Revel et al., 1981). In different cells, the(2'-5') oligo A synthetase activity is in the cytosol (Revel et al.,1981) or in ribosomal salt washes (Dougherty et al., 1980), as well asin the nuclear sap (Nilsen et al., 1982b) and even in large amounts inthe nuclear matrix. It is notable that cellular RNAs can replace poly(rI)(rC) for activation of the enzyme (Revel et al., 1980) and thesynthetase may even have a role in Hn RNA processing (Nilsen et al.,1982a). Some (2'-5') oligo A synthetase is bound to plasma membranes andcan be incorporated in budding virions (Wallach and Revel, 1980). Thesecomplex interactions may ensure a localized action of the (2'-5') oligoA system (Nilsen and Baglioni, 1983) and explain its multiple suggestedroles in normal and virus-infected cells. The synthetase amounts to lessthan 0.1% of the proteins in IFN-treated cells, and its structure couldnot be determined directly.

SUMMARY OF THE INVENTION

The present invention concerns human DNA encoding an enzyme having(2'-5') oligo A synthetase activity. One form of the DNA has thenucleotide sequence set forth in FIG. 7A. Another form of the DNA hasthe sequence of nucleotides 1-1322 set forth in FIG. 7A which overlapswith the sequence of nucleotides 901-1590 set forth in FIG. 7B.

An enzyme having (2'-5') oligo A synthetase activity has the amino acidsequence set forth in FIG. 7A. Another enzyme having (2'-5') oligo Asynthetase activity has the sequence of amino acids 1-364 set forth inFIG. 7A which overlaps with the sequence of amino acids 290-400 setforth in FIG. 7B.

A 1.6 kb and 1.8 kb RNA having nucleotide sequences complementary to thenucleotide sequences in FIGS. 7A and 7B have been isolated.

A method of monitoring the response of a patient to an interferoncomprises measuring the concentration of (2'-5') oligo A synthetase mRNAin cells or body fluids of the patient by hybridizing to the mRNA DNAcomplementary thereto.

Antigenic peptides of the present invention have an amino acid sequencecontained within the amino acid sequences set forth in FIGS. 7A and 7B.Antibodies raised against these antigenic peptides recognize andimmunoprecipitate (2'-5') oligo A synthetase. Also provided areantibodies against all four of the 40 kd, 46 kd, 67 kd and 100 kd formsof (2'-5') oligo A synthetase, as well as antibodies against one ofthese forms which does not cross with the other three forms.

A method of monitoring interferon activity in a subject comprisesmeasuring the amount of (2'-5') oligo A synthetase in a cell or bodyfluid of the subject at predetermined time intervals, determining thedifferences in the amount of said synthetase in the cell or body fluidof the subject within the different time intervals, and determiningtherefrom the amount of synthetase in the cell or body fluid of thesubject and thereby the interferon activity of the subject. Thesynthetase may be measured by contacting the synthetase with an antibodyof the present invention so as to form a complex therewith anddetermining the amount of complex so formed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the structure and sequence of (2'-5') oligo A synthetaseE₁ cDNA clone 174-3:

FIG. 1A depicts the restriction map of E₁ cDNA clone 174-3. The insertbase pairs are numbered in the same direction as pBR322 DNA. The pBR EcoRl site is on the right. Both strands of the insert (dotted lines) weresequenced (Maxam & Gilbert, 1980) from the restriction sites indicatedby the vertical lines. The coding strand is 5' to 3' from right to left.Following the right Pstl site there were 17G and 72T, followed by thedinucleotide GA and the 3T of the sequence shown in (B) which aretherefore not part of the tails. At the 3' end, tails of 45A and 10Cpreceded the left Pstl site.

FIG. 1B depicts the nucleotide sequence having the longest coding frame.The first T is nucleotide 92 following the tails of the insert (rightend in A). The Sau 3A₁ site and the Eco Rl of the insert are atpositions 129 and 480 respectively of the sequence shown.

FIG. 2 depicts the size and induction of E₁ specific mRNAs in SV80 andNamalva cells:

FIG. 2A depicts the hybridization of nick-translated [³² P]-cDNA ofclone E₁ to electrophoretic blots of denaturated poly A⁺ -RNA from SV80cells. The RNAs were prepared at the indicated hour after IFN-beta-1addition. The apparent size of the RNA is indicated on theautoradiography. Left lane, rRNA markers.

FIG. 2B is the same as 2A with RNA from Namalva cells treated withIFN-alpha for the indicated time. Left lane: rRNA markers.

FIG. 3 depicts the characterization by hybridization to RNA blots ofrecombinant plasmid clone C56, harbouring cDNA for an IFN-induced mRNAPoly (A)⁺ RNA from IFN-treated SV80 cells (I) or from non-treated cells(C), 7 micrograms were electrophoresed on agarose gels and afterblotting to nitrocellulose were hybridized to nick-translated [³²P]-plasmid DNA of either the C56 clone, a human HLA cDNA clone or a rattubulin cDNA clone. Exposure was for 48 h. Position of radioactive 18Sribosomal RNA marker is indicated.

FIG. 4 depicts the partial restriction map and nucleotide sequence ofthe C56 450 bp insert. The C56 plasmid was digested with Hine 3,end-labeled with alpha-[³² P]-dCTP by the DNA polymerase I-largefragment (Klenow enzyme, Boehringer) and the Hind 3-Pst 1 fragments wereseparated on a 1% agarose gel. In order to sequence the complementarystrand, the plasmis was 5'-labeled at the Bgl 2 site with gamma [³²P]-ATP by the T₄ -polynucleotide kinase (Biolabs) and the Bgl 2-Pstlfragments were isolated. Sequencing was made by the Maxam and Gilberttechnique. Sequence of coding strand A (right to left) is shown in thelower panel. The two first thymidylic residues of the sequence of strandA probably correspond to the AT tail as indicated in the upper diagram.

FIG. 5 depicts the time course of the induction of C56 mRNA by IFN:

FIG 5A depicts Poly (A)⁺ RNA 7 micrograms from Namalva cells treatedwith IFN-alpha 1000 U/ml for the indicated times were electrophoresed onagarose gels and, after blotting, were hybridized with nick-translated[³² P]-C56 plasmid DNA.

FIG. 5B depicts Poly (A)³⁰ RNA, 7 micrograms from SV80 cells treatedwith 200 U/ml IFN-beta for the indicated times. The asterisk indicatesan RNA sample from cells treated with IFN-beta-1 purified on monoclonalantibody column (2×10⁸ U/mg).

FIG. 5C depicts Poly (A)⁺ RNA, 1 microgram, from SV80 cells treated asin (5B) was hybridized in liquid with 3' end-labeled fragment I of C56DNA (see FIG. 4). The hybrids were treated with S₁ -nuclease andanalyzed on denaturing gels. The mRNA-hybridized probe (→) is shorterthan the self-reassociated probe ( ).

FIG. 6 depicts the restriction map of cDNAs for the 1.6 and 1.8 kb(2'-5') oligo A synthetase mRNAs.

FIG. 6A depicts the map of the 1.6 kb cDNA. The position of the El cDNA(Merlin et al., 1983) and of the lambda gt 10 cDNAs is shown. pA is thepolyadenylation site. The exon limits are shown by vertical dottedlines. The size of the genomic DNA fragments carrying each exon aregiven in parentheses. The vertical arrow shows the position of theadditional splice site in the 1.8 kb RNA. The strategy for sequencingthe 9-21 and 5-21 cDNAs is indicated. The sequence from the 3' EcoRlsite (E) to the Pstl site (P) was determined in the El cDNA (Merlin etal., 1983).

FIG. 6B depicts a map of the 1.8 kb cDNA. The lambda gt10 clone 48-1 wasisolated using the Pstl-Pstl genomic fragment containing exon 8 of the1.8 kb RNA (FIG. 9). Exons are numbered as for the 1.6 kb E cDNA. Thetruncated exon 7 is designated 7a.

FIGS. 7A and B depict the nucleotide sequence of the two (2'-5') oligo Asynthetase cDNAs. The nucleotides of the 1.8 kb cDNA clone 48-1 arenumbered as for the 1.6 kb cDNA clone 9-21. Amino acid numbering isgiven in parentheses. Translation starts at the first or second condonof the ATGATG sequence. Limits between exons are shown by vertical bars.(Glycos.) indicates a possible glycosylation site in E18. Single basevariations, possibly allelic differences, were detected between clonesor genomic DNA in the 1.6 kb sequence at 376 (T for C), 525 (G for A),807 (G for C), 811 (A for G); in the 1.8 kb sequence at 1087 (G for A),1115 (G for C).

FIG. 8 depicts the hydropathy plot of the C-termini of the E16 and E18(2'-5') oligo A synthetases. The computer program of Kyte and Doolittle(1982) was used. Hydrophobic regions are over the midline. The acidicregion in E18 corresponds to amino acids 353 to 358 in FIG. 7.

FIG. 9 depicts the restriction map of the human (2'-5') oligo Asynthetase gene. A map constructed from three overlapping genomic clonesis shown with the position of the 7 exons of the 1.6 kb RNA and theadditional 8th exon of the 1.8 kb RNA (black bars). The insert shows aSouthern blot of genomic DNA with the 48-1 cDNA as probe. Slot 1, DaudiDNA; slot 2, diploid fibroblast FS11 DNA.

FIG. 10 depicts the promoter region of the human (2'-5') oligo Asynthetase gene. A restriction map of the Sphl-Sphl 0.85 kb fragmentfrom the 4.2 kb EcoRl genomic DNA segment in FIG. 9 is shown. The 5' endof the mRNAs is marked as cap.

FIG. 11 depicts the sequence of the human (2'-5') oligo A synthetasepromoter region. The sequence of the Sau3A-Hpal segment of FIG. 10shown, aligned for comparison with the promoter region of the humanIFN-beta-1 gene (Degrave et al., 1981). Numbering is from the presumedcap site. A purine-rich transcription-regulatory sequence around -75 inthe IFN-beta-1 promoter (Zinn et al., 1983), repeated at -10, isunderlined. The TATA box is doubly underlined.

FIG. 12 depicts the SDS-acrylamide gel electrophoresis of ³⁵S-methionine labeled proteins from IFN treated WISH cellsimmunoprecipitated by antiserum to synthetic peptides.

FIG. 13 depicts the expression of E16 cDNA in E. coli. Extracts of E.coli lysogen Agtll-E16 induced by IPTG at 42° C. wee assayed on poly(rI)(rC) agarose beads for (2'-5') oligo A synthesis. Con=extracts of E.coli with E16 cDNA in opposite orientation to lac Z gene. Nam=extractsof IFN-treated Namalva cells. Electrophroresis at pH 3.5 of alkalinephosphatased ³² p-a-ATP labeled products are shown.

FIG. 14 depicts the rapid method for assay of (2'-5') oligo A synthetaseRNAs in human peripheral white blood cells.

FIG. 15 depicts the quick cell blot for (2'-5') oligo A synthetase ERNAs in human PBMC according to the method of FIG. 14. Indicated numberof cells and IFN (16 H treatment) were used. Autoradiography with ³²P-cDNA.

FIG. 16 depicts the (2'-5') oligo A synthetase activity which isadsorbed on anti-B and anti-C IgG-Protein A-Sepharose was measured asdescribed in Example 13. The scheme underneath shows the position ofpeptide B and peptide C in the two (2'-5') oligo A synthetase forms E16and E18 sequenced by Benech et al. (1985b). The blackened area indicatesthe part of E18 which differs from the E16 molecule.

FIG. 17 depicts the electrophoresis and immuno-blotting of extracts fromhuman cells as described in Example 14. The position of the 4 forms of(2'-5') oligo A synthetase is indicated by the numbers on the right ofeach blot. m=¹⁴ C-protein molecular weight markers. IFN treatment isindicated by +.

FIG. 18 depicts electrophoretic immunoblots of extracts from human SV80cells with anti-(2'-5') oligo A synthetase peptide B. Left: crudecytoplasmic extract (Sl.5), cell sap (S100) and microsomes (P100).Right: Na deoxycholate 10% extract of microsomes (DOC-soluble), highsalt wash of microsomes (RWF) and microsomal pellet after saltextraction (Microsomes-KCl).

FIG. 19 depicts the fractionation of S100 and high-salt wash ofmicrosomes (RWF) on DEAE-cellulose and carboxymethyl-cellulose, followedby glycerol gradient. The (2'-5') oligo A synthetase profile and proteindetected by anti-B are shown below the gradients. Electrophoreticimmunoblot shows fraction CM (CM-cellulose eluate from the S100 proteinsnon absorbed to DEAE-cellulose) on the left blot. On the right-hand blotfraction DE (DEAE-cellulose eluate of RWF) and fraction GG (heavy peakof 80-100 kd from glycerol gradient of fraction DE).

FIG. 20 depicts double stranded RNA requirements of various forms of(2'-5') oligo A synthetase from SV80 cells. Fractions are labeled as inFIG. 19. Enzymatic activity measured at indicated concentrations of poly(rI)(rC).

FIG. 21 depicts the results of a radio-immunoassay of (2'-5') oligo Asynthetase with anti-B IgG and ¹²⁵ I-Protein A as described in Example15. Autoradiography is shown. Cells treated for 16 hours with 500 U/mlIFN-βl or left untreated.

FIG. 22 depicts the use of the anti-B for immuno-fluorescencemicroscopic detection of elevated (2'-5') oligo A synthetase levels inlymphocytes from blood of patient with viral disease (middle panel).Right: control with normal serum; left: blood from healthy donor withanti-B strain. (lymphocytes do not stain, only macrophages orgranulocytes give unspecific background).

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns human DNA encoding an enzyme having(2'-5') oligo A synthetase activity and having the nucleotide sequenceset forth in FIG. 7A. The DNA may also comprise the sequence ofnucleotides 1-1322 set forth in FIG. 7A and the overlapping sequence ofnucleotides 901-1590 set forth in FIG. 7B. The DNA of the presentinvention has the restriction enzyme sites set forth in FIG. 9.

An enzyme having (2'-5') oligo A synthetase activity has the amino acidsequence set forth in FIG. 7A. This enzyme comprises about 364 aminoacids and has a molecular weight of about 41,500 daltons. Another enzymehaving (2'-5') oligo A synthetase activity comprises the sequence ofamino acids 1-364 set forth in FIG. 7A and the sequence of amino acids290-400 set forth in FIG. 7B. This enzyme comprises about 400 aminoacids and has a molecular weight of about 46,000 daltons.

The present invention provides a 1.6 kb RNA having a nucleotide sequencecomplementary to the nucleotide sequence set forth in FIG. 7A. Alsoprovided is a 1.8 kb RNA comprising a nucleotide sequence complementaryto the sequence of nucleotides 1-1322 set forth in FIG. 7A and thesequence of nucleotides 901-1590 set forth in FIG. 7B.

A transfer vector of the present invention comprises lambda-gt 11-E16DNA of the present invention, and the lac Z gene, the DNA being fused inphase with the lac Z gene so as to enable expression of the DNA in thesuitable host cell. A microorganism may be transformed by the transfervector. Escherichia coli is a suitable microorganism for thetransformation.

A method of monitoring the response of a patient to an interferoncomprises measuring the concentration of (2'-5') oligo A synthetase mRNAin cells or body fluids of the patient by hybridizing to the mRNA DNAcomplementary thereto. The mRNA may be the 1.6 kb or 1.8 kb RNA of thepresent invention.

A method for evaluating the response of cells and tissues to interferoncomprises hybridizing RNA from cells or tissues exposed to interferonwith cDNA complementary to the RNA, and determining the extent ofhybridization. The RNA is extracted from cells or tissues which havebeen exposed to interferon, immobilized on a membrane filter andhybridized to labeled cDNA specific for interferon-induced mRNAs. Themethod may also comprise in situ hybridization of labeled cDNA to slicesof tissues and then evaluating by microscopic examinationautoradiography, or fluorescence. The cells or tissues analyzed may beof human or other animal origin.

A kit for carrying out a method for evaluating the response of cells andtissues to interferon contains a cDNA complementary to a sequence setforth in FIG. 7A or 7B, reagents to carry out the hybridization testsfor nick-translation with deoxy ribonuclease I and [³² P]-gamma -dCTP,reaches for hybridization on nitrocellulose membranes, and reagents forRNA extraction from cells.

Also provided are antigenic peptides having amino acid sequencescontained within the amino acid sequences set forth in FIG. 7A and FIG.7B.

An antigenic peptide of the present invention has the amino acidsequence comprising the 17 C-terminal amino acids of the amino acidsequence set forth in FIG. 7A and having the amino acid sequence:ARG-PRO-PRO-ALA-SER-SER-LEU-PRO-PHE-ILE-PRO-ALA-PRO-LEU-HIS-GLU-ALS.Another antigenic peptide has the amino acid sequence:GLU-LYS-TYR-LEU-ARG-ARG-GLN-LEU-THR-LYS-PRO-ARG-PRO-VAL-ILE-LEU-ASP-PRO-ALA-ASP.

Antibodies raised against the antigenic peptides of the presentinvention recognize and immunoprecipitate (2'-5') oligo A synthetase. Inone embodiment of the invention, the antibody is specific for all fourof the 40 kg, 46 kd, 67 kd and 100 kd forms of (2'-5') oligo Asynthetase. In one embodiment of the invention, the antibodies areobtained by immunizing an animal with the antigenic peptideGLU-LYS-TYR-LEU-ARG-ARG-GLN-LEU-THR-LYS-PRO-ARG-PRO-VAL-ILE-LEU-ASP-PRO-ALA-ASP.In another embodiment of the invention, the antibody is conjugated witha label, e.g., a fluorescent label, a radioactive label or an enzyme.

This antibody may be used in an assay, e.g., a fluorescent immunoassay,a radioimmunoassay or an enzyme immunoassay, for the 40, 46, 67 and 100kd forms of (2'-5') oligo A synthetase in cells. This assay comprisesincubating the cells with the labeled antibody and detecting cells being(2'-5') oligo A synthetase in any of the 40, 46, 67 and 100 kd forms bydetecting the label. In one embodiment of the invention, the cells aremononuclear blood cells.

The present invention also provides a kit for the detection of all fourforms of (2'-5') oligo A synthetase which comprises the antibody of thepresent invention against the 40, 46, 67 and 100 kd forms of (2'-5')oligo A synthetase.

The invention further provides a 67 kd (2'-5') oligo A synthetaseprotein in a state of enhanced purity. Moreover, a 100 kd (2'-5') oligoA synthetase protein in a state of enhanced purity is also provided.

The present invention additionally provides an antibody against one ofthe 40 kd, 46 kd, 67 kd or 100 kd forms of (2'-5') oligo A synthetasewhich does not crossreact with the other three forms.

A method of monitoring interferon activity in a subject comprisesmeasuring the amount of (2'-5') oligo A synthetase in a cell or bodyfluid of the subject at predetermined time intervals, determining thedifferences in the amount of said synthetase in the cell or body fluidof the subject within the different time intervals, and determiningtherefrom the amount of synthetase in the cell or body fluid of thesubject and thereby the interferon activity of the subject. The amountof synthetase may be measured by contacting the synthetase with anantibody of the present invention so as to form a complex therewith anddetermining the amount of complex so formed.

A method of monitoring interferon activity may further comprise,extracting (2'-5') oligo A synthetase from a cell or body fluid whichhas been exposed to interferon, labeling the extracted synthetase withan identifiable marker to form a labeled synthetase, contacting thelabeled synthetase with an antibody of the present invention undersuitable conditions so as to form a labeled-synthetase-antibody complex,and detecting the marker in the complex, thereby detecting thesynthetase. The marker may be ³⁵ S-methionine.

A kit for carrying out the method of monitoring interferon activitycomprises an antibody of the present invention, materials for extractingthe synthetase, materials for labeling the synthetase, and materials fordetecting the marker and determining the amount of synthetase.

The present invention also provides cloned DNA that specificallyhybridizes to messenger RNAs which appear in human cells after exposureto interferon. The cloned cDNA may be specific for the (2'-5') oligo Asynthetase mRNAs of 3.6, 1.8 and 1.6 kilobase. A cloned DNA of thepresent invention is specific for the mRNA of a 56,000 Mr-protein, whichmRNA is 2 kilobase and which has the sequence defined in FIG. 1.

A partial cDNA clone (El) for the (2'-5') oligo A synthetase mRNA fromhuman SV80 cells, was first obtained through its ability to select byhybridization an mRNA producing 2'-5') oligo A synthetase activity upontranslation in Xenopus laevis oocytes (Merlin et al., 1983). The El cDNAinsert (675 bp) hybridizes to 3 RNA species of 1.6, 1.8 and 3.6 kb whichare coinduced by IFN in SV80 cells, accumulate for 12 hours and arefound in the cytoplasmic polysomal fraction (Benech et al, 1985). Twoother early transcripts (2.7 and 4 kb) appear in lesser amounts.Analysis of various types of human cells has shown that these RNAs aredifferentially expressed in a cell specific manner. In B lymphoblastoidcells (Namalva, Daudi) only the 1.8 kb RNA accumulates, while inamniotic WISH cells, in histiocytic lymphoma U937 cells and in HeLacells, the 1.6 kb RNA is predominantly induced by IFN with some 3.6 kbRNA, but little 1.8 kb RNA. In diploid fibroplasts FSll, in SV80fibroplastoid cells and in the T cell line CEMT, all 3 stable RNAs areexpressed (Benech et al., 1985). The type of (2'-5') oligo A synthetaseRNA expressed does not depend on the species of IFN used (alpha, beta,or gamma) but rather seems developmentally regulated in the cell.

The different (2'-5') oligo A synthetase transcripts appear to originatefrom a single gene (Benech et al., 1985). Restriction mapping showed: 1)that the El cDNA corresponds to the 3' end of the 1.6 kb RNA; 2) thatthe 1.8 kb RNA has a different 3' end than the 1.6 kb RNA and containsan additional downstream exon; and 3) that the 3.6 kb RNA has the same3' end as the 1.8 kb RNA but is incompletely spliced.Hybridization-translation experiments using specific genomic DNAfragments also demonstrate that both the 1.8 and 1.6 kb RNAs activelycode for (2'-5') oligo A synthetase (Benech et al., 1985).

cDNA clones for the 1.6 and 1.8 kb RNAs have been isolated andsequenced, which enabled the deduction of the amino acid sequences oftwo forms of the IFN-induced (2'-5') oligo A synthetase in human cells.The two proteins differ in their C-termini, which is hydrophobic in the1.6 kb RNA product (E16) and acidic in the 1.8 kb RNA product (E18). Acomplete mapping of the (2'-5') oligo A synthetase gene shows that the1.6 kb RNA is coded by 7 exons and the 1.8 kb RNA by 8 exons. Thesequence of the presumed transcription initiation site and promoterregion of the IFN-activated human (2'-5') oligo A synthetase gene showsa striking homology to the promoter region of the human IFN-beta-1 gene.

Two peptide sequences were chosen from the total amino acid sequences ofE16 and E18 to serve as antigens for the induction of antibodies againstthe active (2'-5') oligo A synthetase molecule. Peptide B, having thesequence:

    GLU-LYS-TYR-LEU-ART-ARG-GLN-LEU-THR-LYS-PRO-ARG-PRO-VAL-ILE-LEU-ASP-PRO-ALA-ASP

comprises amino acids 284 to 303 common to both the E18 and the E16sequences (Benech et al., 1985b). Peptide C, having the sequence:

    ARG-PRO-PRO-ALA-SER-SER-LEU-PRO-PHE-ILE-PRO-ALA-PRO-LEU-HIS-GLU-ALA

comprises the C terminus of E16, i.e., residues 348 to 364.

Both peptides were synthesized by the solid-phase peptide synthesismethod of Barany and Merrifield (1980). After purification on SephadexG25 columns in 2 M acetic acid, the peptides were linked to KeyholeLimpet Hemocyanin (Calbiochem). Fsterification of the NH² -terminalarginine of peptide C with p-aminophenylacetic acid allowed tocovalently link the peptide to the carrier protein through itsamino-terminus (Spirer, et al., 1977). Peptide B was coupled to thecarrier protein by ethylene diamine carbodiimide (Hoare and Koshland,1967).

Rabbits were injected subcutaneously with 1 mg carrier-coupled peptide(equivalent to 0.2 mg pure peptide) which was emulsified in completeFreund's adjuvant. Rabbits were boosted twice at two week intervals with0.5 mg of carrier-coupled peptide in incomplete adjuvant. Subsequentboosting was done by injection of 0.1 mg carrier-free peptide inincomplete adjuvant, and was continued until maximal antibody response.The titer of antibodies in the rabbit sera were measured inenzyme-linked immunosorbent assays (Green, et al., 1982) using thecarrier-free peptides.

The fibroblastoid cell line SV80 and the amniotic cell line Wish weregrown to confluent monolayers on plastic dishes and the Daudi cell linewas grown in suspension to 1.5×10⁶ cells/ml (Benech, et al., 1985a).Cultures were treated for 16-24 hours with rIFN-β1, 500 U/ml. The humanrIFN-β1 was produced by genetically engineered CHO cells and purified tohomogeneity by monoclonal antibody affinity chromatography(Chernajovsky, et al, 1984).

Cells were washed twice with phosphate buffered saline (PBS) at 4° C.and lysed in the cold in Buffer A (20 mM Hepes buffer, pH 7.5, 5mMmagnesium acetate, 30 mMβ-mercaptoethanol, 100 μM phenylmethyl sulfonylfluoride (PMSF), 10% glycerol and 0.5% Nonidet P-40 (NP40)). Nuclei andunbroken cells were eliminated by centrifugation at 1,500 γ for 10minutes. The supernatant (Sl.5) was centrifuged 10 minutes at 15,000 γin an Eppendorf Microfuge to obtain a mitochondria and lysosome-freesupernatant (Sl.5). Protein concentrations were measured by microassays(Bradford, 1976).

Centrfigutation of S15 for 2 hours at 100,000 γ in a Beckmanrefrigerated ultracentrifuge was used to prepare cell sap (S100) andmicrosome (P100) fractions Alilquots of S15 containing 1-2 μg proteinwere incubated in 20 μl reactions containing 25 mM Hepes buffer, pH 7.5,20 mM magnesium acetate, 1 mM dithiothreitol, 1.5mM ATP, 4 μCi of ³²P-α-ATP, 50 μg/ml poly(rI)(rC) (from PL-Biochemicals) for 2 hours at 30°C. After boiling for 5 minutes and microfuge centrifugation, bactericalalkaline phosphatase was added at 25 U/ml to an aliquot and the reactionincubated for 2 hours at 37° C. From 2 to 7 μl were spotted on Whatman 3MM paper and analyzed by electrophoresis at 3,000 V in pyridine/aceticacid, pH 3.5. After autoradiography, the (A2'p)nA oligomer spots werecut out and counted.

Aliquots of crude cellular factions (30 μg protein) were adjusted withLaemmli's sodium-dodecyl-sulphate-polyacrylamide gel electrophoresisloading buffer (Laemmli, 1970) and boiled 10 minutes beforeelectrophoresis on 7.5 or 10% gels. Amersham's ¹⁴ C-methylated proteinswere used as molecular weight standards (10⁴ cpm) Electrophoretictransfer onto nitrocellulose paper (Schleicher and Schuell BA85) wascarried out in 25 mM Tris-base, 192 mM glycine and 20% methanol. Theblots were preincubated in 0.9M NaCl, 0.01M Tris-HCl, pH 7.5, 10% (v/v)of a 1% fat milk solution, 10% (v/v) heat-inactivated fetal calf serumand 0.05% Tween-20, either for 2 hours at 37° C. or overnight at 4° C.followed by 30 minutes at 37° C. Blots were then incubated withanti-(2'-5') oligo A synthetase peptide B antibodies in form of rabbitIgG 0.01 mg/ml, for 2 hours at 37° C. Blots were washed 5 times for 10minutes in 4% fetal calf serum and incubated in the complete abovepreincubation mixture containing 10⁶ cpm/ml of 125I-protein A (Amersham,30 mCi/mg}for 1 hour at 37° C. Blots were washed and subjected toautoradiography.

First aliquots of 5-10 μl anti-(2'-5') oligo A synthetase peptide Brabbit serum were adsorbed on 3mg of Protein A-Sepharose (Pharmacia)equilibrated in PBS with 3% bovine serum albumin (BSA), for 30 minutesat room temperature, then washed with PBS-1% BSA. Forimmunoprecipitation of the (2'-5') oligo A synthetase enzymaticactivity, aliquots of 2μg of S15 proteins in a final volume of 20μl ofbuffer A were adsorbed on the above pelleted IgG-Protein A Sepharose for2 hours at 4° C. The suspension was diluted 5 fold in Buffer A and thesupernatant transferred to another tube. The pellet was washed 3 timeswith 0.5 ml Buffer A and was then suspended in 25 μl of the enzymereaction mixture (see above). Activity was measured also on aliquots ofthe non-bound supernatant.

For labeling (2'-5') oligo A synthetase, Wish cells were grown toconfluent monolayers on 3 cm plastic dishes and treated for 12 hourswith 500 U/ml rIFN-βl. The medium was replaced by 0.5 ml methionine-freeDMEM (GIBCO) containing 500 μCi of ³⁵ S-methionine (Amersham 400mCi/mmol) and cells incubated for 2 hours, washed with PBS andhomogenized in Buffer A. The S15 was used for immunoprecipitation. About10⁷ cpm of S15 proteins were added to the pelleted Protein A-Sepharoseand mixed for 2 hours at 4° C. The beads were washed with 1% BSA, 1%NP40, 2M KCl in PBS and twice with PBS only. Samples were analyzed bysodium-dodecyl-sulphate-polyacrylamide gel electrophoresis.

EXAMPLE 1 Measure of (2'-5') oligo A synthetase mRNA by cDNA clones A)Isolation of E-cDNA clones

Total RNA was prepared from SV80 cells (SV40-transformed humanfibroblasts) treated for 12 hours with 200 units per ml IFN-beta. TheRNA was extracted by 3M LiCl - 6M urea and purified by passage on oligodT-cellulose. The 0.4 mg poly A⁺ -RNA obtained were fractionated in apreparation of gel electrophoresis apparatus in 1.5% agarose/6M urea 25mM sodium citrate pH 3.5. The 17-18S RNA fraction, was used to preparecDNA as follows: 2 micrograms RNA were heated for 1 min at 90° C. with 2micrograms oligo (dT)₁₂₋₁₈ in 60 microliters water, cooled at 0° C.,supplemented with salts to a final concentration of 50 mM Tris-HCl pH8.3, 10 mM MgCl₂, 75 mM KCl and incubated 5 min at 42° C. before adding1 mM dithiothreitol, 1 mM each dATP, dIGP, 0.5 mM dCTP, 20 micro-Ci ³²P-dCTP (300 (Ci/mmol)), 4 mM Napyrophosphate and 20 units reversetranscriptase in a final volume of 0.1 ml. The mixture was incubated for45 minutes at 42° C., the reaction was stopped with 10 mM EDTA, 0.2%Na-dodecyl sulfate and the cDNA extracted with phenol-chloroform,treated with 0.3N NaOH for 2 hours at 52° C. and neutralized. The cDNAwas filtered on Sephadex G-75, ethanol precipitated, and tailed by dATPwith terminal transferase. The synthesis of the second cDNA strand wasprimed with oligo (dT) and carried out as for the first strand for 2hours at 42° C. but without radioactive nucleotide and withoutpyrophosphate. To insure blunt ends the ds cDNA was incubated with E.coli DNA polymerase I large fragment first in 20 mM Tris-HCl pH 8, 75 mMKCl, 5 mM MgCl , 1 mM dithiothreitol for 5 minutes at 37° C. (trimmingreaction) and then under the conditions of filling-in with ATP. The dscDNA was fractionated by sedimentation on a 5-20% sucrose gradient andthe heaviest fractions were tailed with dCTP and annealed with equimolaramounts of Pstl-cut pBR322 plasmid DNA tailed with dCTP. About 7 ng DNAwere mixed with 100 microliters of frozen, CaC12-treated, E. coli MM294.After 30 minutes at 0° C., and 5 minutes at 37° C., the bacteria weregrown in 2ml of LB-broth for 2 hours at 37° C., and plated on LB-agarplates with 10 micrograms/ml tetracycline. About 1.4×10⁵tetracycline-resistant, ampicillin-sensitive colonies were obtained permicrogram recombinant plasmid DNA.

To identify the cDNA clone of the (2'-5') oligo A synthetase mRNA, atotal of 3,000 plasmid DNA clones were screened by hybridization to RNAof IFN-treated SV80 cells, and the DNA-selected RNA was tested byinjection into Xenopus laevis oocytes and a measure of the (2'-5') oligoA synthetase activity formed according to the method of Shulman andRevel (1980). Pools of plasmid DNA from 12 individual clones (3micrograms DNA each; cut with Eco Rl) were applied onto a 0.4 cmdiameter nitrocellulose filter and prehybridized for 2 hours at 37° C.in 50% formamide, 2 mM Pipes buffer pH 6.4, 0.75M NaCl, 1 mM EDTA(buffer A). Three filters with pBR322 DNA and thirty filters ofrecombinant DNA pools were incubated together with 300 micrograms polyA⁺ -RNA [calculated to have a 10-fold excess of each insert cDNA overthe presumed amount of (2'-5') oligo A synthetase mRNA, 0.09 microgramsor 0.03%] in 1 ml buffer A for 20 hours at 37° C. The filters werewashed twice at 37° C. with buffer A, 4 times in 20 mM Tris-HCl pH 7.5,0.15M NaCl, 1 mM EDTA, 0.5% Na dodecyl sulfate (once at 37° C. and 3times at 52° C.) and then 4 times with 10 mM Tris-HCl pH 7.5, 1 mM EDTA(buffer C) at 52° C. Each filter was next washed individually in bufferC at 52° C. and the RNA was eluted by heating 2 min at 96° C. in 0.3 mlbuffer C with 40 micrograms rabbit liver tRNA per ml. After quickcooling the ethanol precipitation, the RNA was dissolved in 2microliters water. Ten Xenopus laevis oocytes were microinjected with0.7 microliters RNA and after 18 hours at 19° C., the oocytes werehomogenized in their incubation medium (Shulman and Revel, 1980) and0.15 ml of homogenate was mixed with poly (rI) (rC)-agarose beads. Thebeads were incubated for 16 hours at 30° C. with 2.5 mM [³² P]-alpha-ATP(0.3 Ci/mmol), 10 mM dithiothreitol, and 10 microliters of the liquidphase were incubated with 0.35 units bacterial alkaline phosphatase in30 mM Tris-base for 60 minutes at 37° C. The idgest was submitted topaper electrophoresis on Whatman 3 MM paper at 3,000 V for 4 hours, andthe spots corresponding to (2'-5') ApA and (2'-5') ApApA were cut andcounted by scintillation. From the DNA-selected RNA, 1 microliter wasused for in vitro translation in a reticulocyte lysate as described inWeissenbach et al. (1979), to measure the total mRNA activity of thesample.

The ratio of (2'-5') oligo A synthetase activity over total mRNAactivity in the DNA-selected RNA samples was calculated for each filter.One filter with pool 174, out of the 250 pools of 12 individualplasmids, gave consistently a ratio about 10 times higher as other poolsor as pBR322 DNA. The plasmid DNA of each individual clone of pool 174was tested on separate filters and clone 174-3 was found to giveconsistently a 35-100 fold enrichment of the (2'-5') oligo A synthetasemRNA over total mRNA as compared to total RNA or pBR322 DNA-selected RNA(Table 1). Clone 174-3 was identified as the (2'-5') oligo A synthetasecDNA and designated E-cDNA. The structure and sequence of this cDNA isshown in FIG. 1. The E-cDNA clone contains the sequence for the 100carboxy terminal amino acids of the enzyme and a 192 nucleotide-longuntranslated region preceding the poly A-tail.

                                      TABLE 1                                     __________________________________________________________________________    IDENTIFICATION BY HYBRIDIZATION-TRANSLATION OF THE CLONE                      OF (2'-5') OLIGO A SYNTHETASE cDNA                                            E mRNA activity measured by oocyte injection (2) of induced RNA                           Expt. 1   Expt. 2   Expt. 3                                                   (2'-5')   (2'-5')   (2'-5')                                                   oligo A                                                                            (specific                                                                          oligo A                                                                            (specific                                                                          oligo A                                                                            (specific                                            cpm  activity)*                                                                         cpm  activity)*                                                                         cpm  activity)*                               __________________________________________________________________________    Total poly A.sup.+ -RNA                                                                   4050 (0.007)                                                                            3440 (0.004)                                                                            4900 (0.01)                                   RNA selected on:                                                                          350       570  (0.03)                                                                             670  (0.02)                                   pBR filters 625  (0.05)                                                                             595       725                                           plasmid pool 174                                                                          2320      --        --                                            other pools  230**                                                                             ±60                                                                             --        --                                            Clones of pool 174:                                                                      1                                                                              560       700       995                                                      2                                                                              625       950  (0.34)                                                                             825                                                      3                                                                              6460 (4.78)                                                                             39,800                                                                             (2.7)                                                                              11,235                                                                             (0.75)                                              4                                                                              600       1,820                                                                              (0.48)                                                                             1,030                                                                              (0.03)                                              5                                                                              745       500  (0.49)                                                                             530                                                      6                                                                              985       475       630                                                      7                                                                              1270 (0.1)                                                                              365       605                                                      8                                                                              395       800  (0.02)                                                                             600                                                      9                                                                              490       100       1,030                                                                              (0.03)                                             10                                                                              1465 (0.14)                                                                             290       1,155                                                                              (0.06)                                             11                                                                              1860 (0.27)                                                                             365       735                                                     12                                                                              540       320       915                                           No RNA      195       185       590                                           __________________________________________________________________________     *Specific activity ratio of (2'-5') oligo A synthesis or mRNAinfected         oocytes to translation of same RNA in reticulocyte lysates.                   **Average of 28 pools.                                                   

B) Measure of interferon-induced (2'-5') oligo A synthetase mRNA byhybridization of E-cDNA

Plasmid DNA of clone 174-3 (E-cDNA) can be used to detect thecomplementary RNA on electrophoretec blots of total cell RNA. Poly A³⁰-RNA is prepared from SV80 cells treated various times by 2000/mlinterferon-beta and 7 micrograms RNA are denatured in 50% formamide, 6%formaldehyde, electrophoresed in 1.3% a9arose with 6% formaldehyde andblotted onto nitrocellulose according to the procedures of Thomas (1980)and Fellous et al. (1982). The E-cDNA plasmid labeled bynick-translation with according to Merlin et al. (1983), is hybridizedto the nitrocellulose blot.

In SV80 cells, three RNA species which are all coordinately induced bythe interferon treatment, hybridize with E-cDNA (FIG. 2), a large RNAspecies of 3.6 kilobases and 2 smaller species of 1.85 and 1.65kilobase. In non-treated SV80 cells, no E-specific RNA is found. The 3RNA species appear at 4 hours, are maximum at 12 hours and decreaseslowly thereafter. The RNAs are still clearly detected at 24 hours afterinterferon. Additional RNA species seen only at 4 hours are mostprobably precursors of the more stable species. The same 3 RNA speciesare seen in human diploid fibroblasts treated by interferon. However, incells of the hemopoietic lineage such as lymphoblastoid Namalva cells,only one main RNA species hybridizes to E-cDNA (FIG. 2) and correspondsto the 1.85 kilobase RNA species. The same RNA pattern is seen in otherlymphoblastoid cells, in erythroid HL-60 and in promonocyte U937 cells.

The different E-specific RNA pattern in fibroblasts and lymphoid cellscorresponds to different forms of the (2'-5') oligo A synthetase inthese cells. Lymphoid cells contain an enzyme of molecular weight 30,000daltons, while fibroblasts contain two forms of the enzyme of molecularweight 80,000 and 30,000 daltons, as reported by Revel et al. (1982).The small 1.85 kilobase mRNA is sufficiently long to code for the 30,000Mr enzyme but not for the larger form, while the 3.6 kilobase E-mRNAcodes for the 80,000 Mr form of the enzyme. All three E-specific RNAspecies hybridize to a single clone of human genomic DNA, and probablyoriginate from a single gene, the 3.6 kilobase RNA having an additionalinterferon exon as compared to the 1.85 kilobase RNA.

Leucocyte interferon-alpha induces E-specific RNA as well as doesfibroblast interferon-beta. The multiplicity of RNA species revealed byhybridization to EcDNA suggests that different interferon species, whichall induce (2'-5') oligo A synthetase, could induce different forms ofthe RNA and of the enzyme. Different interferon species can also vary intheir efficacy for inducing E-mRNA.

RNA to be treated in the above hybridization assay to E-cDNA may beprepared from various cells in culture or from tissues taken frompatients receiving interferon therapy or suffering from viral diseasesor from disease in which an elevated (2'-5') oligo A synthetase wasobserved (Schattner et al., 1981). RNA may also be prepared from bloodcells, such as leukocytes, obtained from peripheral blood. Theelectrophoretic blot can be replaced by a dot-hybridization method, inwhich RNA samples are directly applied to nitrocellulose in circles orrectangles of defined area, and the radioactive cDNA is hybridized tothe nitrocellulose sheet. The radioactivity of each circle of rectangeis then measured by direct counting or by autoradiography followed byscreening of the autoradiographic film.

An alternative method is to perform hybridization in situ on tissueslices obtained from biopsies of tissues exposed to interferon. This canbe preferentially applied to brain biopsies in patients receivinginterferon for a brain viral disease or tumor, in order to measurewhether the brain has been exposed to interferon when the drug is giveneither by intrathecal injection or by systemic injection. The method maybe applied to skin biopsies when the interferon treatment is givenlocally as an ointment for skin lesions. It is obvious that many otherapplication are possible. The tissue slices may be fixed and hybridizedin situ to radioactive DNA, followed by an autoradiography with asensitive photographic emulsion. The cDNA may also be labeled byfluorescent nucleotides or by modified nucleotides which can bindfluorescent molecules, and the hybridization to the tissue slice can bemonitored by fluorescent microscopy.

An increase in hybridization of the E-cDNA was compared to a propercontrol cell RNA or tissue sample, indicating that the cell or tissuehas been exposed to interferon. The rapidity (4-24 hours) andsensitivity (1-200 units of interferon per ml) of the method makes itvery useful to follow a treatment by external interferon, or formationof endogenous interferon in blood and tissue of patients.

EXAMPLE 2 Cloned cDNA for the interferon-induced 56,000 Mr protein A)Isolation of cloned C56-cDNA

The cloned cDNA was isolated from the library of recombinant plasmidsdescribed in Example 1. The principle of the method used wasdifferential hybridization. Two duplicate sets of the 3,000 bacterialclones grown on nitrocellulose filters were hybridized either to [³²P]-cDNA from 17S-18S poly A⁺ -RNA of SV80 cells treated byinterferon-beta(200 U per ml), or to [³² P]-cDNA from total poly A⁺ -RNAof non-treated SV80 cells. The radioactive cDNA were reverse transcribedfrom mRNA as in Example 1. About 40% of the bacterial clones hybridizedstrongly to the "interferon-treated" cDNA probe and 8% 9ave a cleardifferential signal, hybridizing preferentially or uniquely to the"interferon-treated" cDNA as compared to the "non-treated" cDNA. Thelatter group of clones was then screened by hybridizing the plasmid DNAfrom each clone, labeled radioactively by nick-translation, toelectrophoretic blots of RNA from interferon-treated SV80 cells and fromnon-treated cells. By this criterion, 1-2% of the original 3,000bacterial clones were found to contain a plasmid cDNA clonecorresponding to an interferon-induced mRNA. One of these plasmid cDNAclones, designated C56, showed a particularly strong differentialhybridization. This C56 DNA hybridizes to an 18S RNA present ininterferon-treated cells but completely absent from control cell RNA(FIG. 3). In comparison, HLA-A,B,C mRNA which is increased 5-fold inSV80 cells after interferon-treatment (Fellous et al., 1982), appearsmuch less induced than C56 mRNA and under the experimental conditions ofFIG. 3, gives a clear signal also with "non-treated" RNA.

The mRNA selected by hybridization to C56 cDNA immobilized onnitrocellulose filters, followed by elution from the films (as inExample 1) was translated in a reticulocyte lysate cell-free system andthe [³⁵ S]-methionine-labeled translation products were analyzed bypolyacrylamide gel electrophoresis in Na-dodecyl sulfate according tothe method described in Weissenbach et al. (1979) adapted from Laemle(1970). The C56 cDNA-selected RNA is translated into a 56,000-Mrprotein. The sequence of the C56 cDNA permits one skilled in the art todeduce 65 amino acids of the carboxy terminal sequence of the 56,000 Mrprotein (FIG. 4).

Hybridization of the C56 cDNA to RNA extracted from SV80 cells treatedvarious times by interferon-beta (200 U per ml), shows that the C56 mRNAstarts to appear at 1 hour after interferon addition (FIG. 5). The C56RNA reaches its maximum after 4 hours, but is still detectable, althoughreduced, at 24 hours. Induction of C56 mRNA was also demonstrated indiploid fibroblasts, and in lymphoblastoid cells. Induction wasproportional to the concentration of interferon between 10 and 200 unitsper ml. C56 mRNA was also induced by interferons alpha and gamma,although the latter was less efficient. The absence of this mRNA innon-treated cells and its strong and rapid increase after interferonaddition make the C56 cDNA an excellent probe to evaluate the responseof cells to interferon. The techniques described for E-cDNA in Example1, can be similarly applied to the C56 cDNA.

The availability of a number of cDNA corresponding to mRNA induced byinterferon offers new perspectives. In particular, interferon- is neededat 100-fold lower concentrations to induce HLA-A,B,C mRNA than toinduced E-mRNA or C56 mRNA (Wallach et al. (1982); on the other hand,some subspecies of interferon-alpha, such as alpha-d can induce E-mRNAwhen a concentration 100 times lower than those needed to induceHLA-A,B,C mRNA. A comparison of the hybridization of different clonedcDNAs to the same RNA sample, can indicate what type of interferon isinvolved. Thus, more information can be derived from the comparison ofdifferent cDNA than from the use of only one cDNA probe.

EXAMPLE 3 A Kit For The Measure Of Interferon-Induced mRNAs

The Kit would provide the cloned cDNA specific for the mRNA of the(2'-5') oligo A synthetase and for the mRNA of the 56,000 Mr protein,described herein, as well as reagents to carry out the hybridizationtests: comprising reagents for nick-translation with deoxy-ribonucleaseI and -gamma-dCTP, [³² P]-gamma-dCTP, reagents for hybridization onnitrocellulose membranes, and reagents for RNA extraction from thecells.

EXAMPLE 4 Sequence of cDNA for the 1.6 kb (2'-5') oliqo A synthetasemRNA

The partial E₁ cDNA clone (Merlin et al., 1983), shown to be the 3' endof the 1.6 kb (2'-5') oligo A synthetase by IFN in human cells (Benechet al, 1985) was used to screen a lambda gt10 cDNA library from SV80cell RNA (Wolf and Rotter, 1985). By restriction mapping, clone lambdagt10 9-2 was found to contain the El cDNA at the 3' end of a 1.32 kbEcoRl insert (FIG. 6A) which was subcloned in pBR (9-21 cDNA).Sequencing was carried out as outlined in FIG. 6A and confirmed that thethe 9-21 cDNA contains the C-terminus and 3' untranslated sequencepreviously reported for the E₁ cDNA (Merlin et al., 1983). The 9-21 cDNAsequence (FIG. 7) predicts an open reading frame of 364 amino acidsstarting at an AIGATG sequence. A computer program based on the 3-baseperiodicity of protein-coding sequences (Trifonov, 1984) indicated thatthe only compatible reading frame is the one starting from this ATGATG.It is possible that translation initiates at the second ATG in thissite, since it is the only one preceded by an A at -3 and havinghomology with the concensus translation initiation sequence (Kozak,1984).

The enzyme thus coded by the 1.6 kb (2'-5') oligo A synthetase RNA has amolecular weight of about 41,700 daltons, based on the deduced aminoacid sequence, which is in good agreement with the apparent 38,000 Mrprotein seen by SDS-polyacrylamide gel electrophoresis of the in vitrotranslation product of RNA hybridized to E₁ cDNA (Merlin et al., 1983).The C-terminal heptadecapeptide predicted by the open reading frame, wassynthesized chemically and used to immunize rabbits. The antiserumobtained (C in FIG. 12) precipitates specifically a protein migrating at38,000-Mr in SDS gel electrophoresis from ³⁵ S-methionine labeledextracts of cells treated by IFN which is absent from untreated cells.Two experiments confirmed that this protein has (2'-5') oligo Asynthetase activity: 1) it was removed from the extracts by passsagethrough a poly (rI)(rC) agarose column; and 2) the supernatant remainingafter immunoprecipitation was depleted of a large part of the enzymaticactivity.

EXAMPLE 5 Sequence of cDNA for the 1.8 kb (2'-5') oliqo A synthetasemRNA

A genomic DNA fragment corresponding to the additional exon of the 1.8kb RNA (Benech et al., 1985; see FIG. 9) was used as probe to isolate anE18 cDNA clone, 48-1, from the same lambda gt10 cDNA library of SV80RNA. The restriction map of the E18 cDNA clone (FIG. 6B) confirmed thatits 5' end is part of the E16 cDNA but that its 3' end differs.Sequencing (FIG. 7) revealed that the junction is at nucleotide 1071 ofthe E16 9-21 cDNA clone, the last 247 nucleotide of E16 being replacedby a 515 nucleotide-long sequence terminated by a differentpolyadenylation site. This difference accounts for the 0.2 kb differencein size between the two mRNAs seen on Northern blots. The 5' portion ofthe E18 cDNA shows no base change from the sequence of the E16 cDNA, butis incomplete. The gene mapping described below, indicates that both 1.6and 1.8 kb mRNAs have the same 5' end.

The 3' region of the E18 cDNA which diverges from the E16 sequence,contains an open reading frame ending after 54 codons. This readingframe, which leaves a 50 nucleotide-long untranslated region, wasconfirmed by the computer program based on the 3 base periodicity ofprotein-coding sequences (Trifonov, 1984). An alternate longer openreading frame would not be in the same computed phrase as the 5' portioncommon with the E16 cDNA. A hydropathy plot (Kyte and Doolittle, 1982)on the prprediced C-termini of the 1.6 and 1.8 kb mRNA protein products,indicates a striking difference between the two forms of the (2'-5')oligo A synthetase (FIG. 8). The C-terminus of the E16 protein is veryhydrophobic, while that of the E18 protein is hydrophilic and containstwo acidic regions (Asp-Asp-Glu-Thr-Asp-Asp and Glu-Glu-Asp) (FIG. 7).Furthermore, a possible glycosylation site is present in the C-terminusof the E18 product (FIG. 7).

The 9-21 cDNA was subcloned in lambda-gtll so as to fuse the codingframe in phase with the lac Z gene. Extracts of the E. coli lysogencontaining this phase, showed clearly (2'-5') oligo A synthetaseactivity after binding to poly(rI)(rC) agarose, while no activity wasfound when the 9-21 cDNA had been fused in the opposite orientation(FIG. 13). This expression in E. coli demonstrates that the cDNA indeedcorresponds to the structural gene coding for the ds RNA activated(2'-5') oligo A synthetase and that the protein of about 40 kd coded bythe IFN induced RNA is the enzyme itself, and not a regulatory factor.This protein does not seem to require post-translational modificationsto exhibit enzymatic activity.

The transformed cell containing the 9-21 cDNA has been designatedEscherichia coli lambda-gtll-E16 and deposited under Accession No. I496with the Collection National Cultures de Micro-organismes, InstitutPasteur, 25 rue du Docteur Roux, 75724-Paris-Cedex 15, France. Thisdeposit was made pursuant to the Budapest Treaty On the InternationalRecognition Of The Deposit Of Microorganisms For The Purposes Of PatentProcedure.

EXAMPLE 6 Organization of the human (2'-5') oligo A synthetase gene

Three overlapping genomic clones were isolated using the E₁ cDNA asprobe (Benech et al., 1985), one from a library of partial EcoRl digestof human blood cell DNA (Mory et al., 1981) and two from a library ofpartial Alul and Hae3 digest of embryonic human DNA (Maniatis et al.,1978). The genomic clones represent about 29 kb of human DNA and noevidence for more than one E gene was found while screening thelibraries. Southern blots of genomic DNA are consistent with theexistence of a single gene (FIG. 9). By Northern blot analysis usinggenomic DNA fragments as probes, by Sl nuclease mapping and bysequencing, the E16 cDNA 9-21 was shown to correspond to five exons onthe gene (FIG. 9). The ATGATG sequence is found in exon 3, while thetermination codon and 3' untranslated region with the polyadenylationsite of the 1.6 kb RNA are found in exon 7. The structure of the more 5'exons 1 and 2 is described below. The sequences of the intron-exonboundaries were determined (Table 2) and follow the CAG and GT rule forthe splice acceptor and donor sites (Breathnach and Chambon, 1981).

A sequence CTGAC/T is commonly found not far from the splice acceptor,as reviewed recently by Keller (1984). It is notable that the CTGAC/Tregion shows base complementarity to the sequence of the intron/exon 3'boundary (acceptor site; Table 2), in addition to the complementarity ofthe intron donor site with the CTGAC sequence pointed out by Keller(1984) as playing a role in the lariot model.

The sequences of the 5 exons containing the coding region of the (2'-5')oligo A synthetase produced by the 1.6 kb mRNA, indicates that theenzyme is composed of domains with differing amino acid compositions(Table 3}. The first exonic domain (60 amino acids) is rich in asparticacid, in the second (amino acids 61 to 156) arginine is predominant, thenext two exons (amino acids 157 to 218 and 219 to 295) are lysine rich,and the C-terminus of the E16 product (296 to 364) is very rich inproline and alanine.

                                      TABLE 2                                     __________________________________________________________________________    EXON-INTRON BOUNDARIES IN THE HUMAN                                           (2'-5') OLIGO A SYNTHETASE GENE                                               __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                     __________________________________________________________________________     For exon numbering see FIGS. 7 and 9. The selfcomplementary regions           between the CTGAT/C, or CTTAC, CTGTC (Keller, 1984) and splice acceptor       CAG are underlined. The polyadenylation sites with a conserved                undecanucleotide of the 1.6 and 1.8 kb RNAs (see FIG. 7) are underscored      by dots.                                                                      ##STR7##                                                                      The start and end of each exon is numbered as in the 921 E cDNA of FIG. 7

                                      TABLE 3                                     __________________________________________________________________________    EXONIC DOMAINS OF THE E16 AND E18                                             (2'-5') OLIGO A SYNTHETASES                                                                                E16 C-term.                                                                          E18 C-term.                                   1-60 61-156                                                                             157-218                                                                            219-295                                                                            296-346                                                                            347-364                                                                              347-400                                   AA  (60) (96) (62) (77) (51) (18)   (54)                                      __________________________________________________________________________    ALA 2 (3.3)                                                                            7 (7.3)                                                                            0 (0.0)                                                                            3 (3.9)                                                                            4 (7.8)                                                                             3 (16.7)                                                                            4 (7.4)                                   ARG 4 (6.7)                                                                            10 (10.4)                                                                          3 (4.8)                                                                            5 (6.5)                                                                            1 (2.0)                                                                            1 (5.6)                                                                              2 (3.7)                                   ASH 1 (1.7)                                                                            2 (2.1)                                                                            2 (3.2)                                                                            3 (3.9)                                                                            3 (5.9)                                                                            0 (0.0)                                                                              1 (1.9)                                   ASP  6 (10.0)                                                                          5 (5.2)                                                                            2 (3.2)                                                                            1 (1.3)                                                                            4 (7.8)                                                                            0 (0.0)                                                                              5 (9.3)                                   CYS 4 (6.7)                                                                            1 (1.0)                                                                            2 (3.2)                                                                            2 (2.6)                                                                            1 (2.0)                                                                            0 (0.0)                                                                              1 (1.9)                                   GLN 1 (1.7)                                                                            7 (7.3)                                                                            6 (9.7)                                                                            5 (6.5)                                                                            2 (3.9)                                                                            0 (0.0)                                                                              3 (5.6)                                   GLU 2 (3.3)                                                                            7 (7.3)                                                                            5 (8.1)                                                                            4 (5.2)                                                                            2 (3.9)                                                                            1 (5.6)                                                                              5 (9.3)                                   GLY 2 (3.3)                                                                            9 (9.4)                                                                            3 (4.8)                                                                            3 (3.9)                                                                             6 (11.8)                                                                          0 (0.0)                                                                              2 (3.7)                                   HIS 1 (1.7)                                                                            0 (0.0)                                                                            1 (1.6)                                                                            1 (1.3)                                                                            0 (0.0)                                                                            1 (5.6)                                                                              3 (5.6)                                   ILE 5 (8.3)                                                                            2 (2.1)                                                                            3 (4.8)                                                                            4 (5.2)                                                                            2 (3.9)                                                                            1 (5.6)                                                                              2 (3.7)                                   LEU 5 (8.3)                                                                            13 (13.5)                                                                           8 (12.9)                                                                          10 (13.0)                                                                           6 (11.8)                                                                           2 (11.1)                                                                            2 (3.7)                                   LYS 5 (8.3)                                                                            2 (2.1)                                                                             7 (11.3)                                                                           9 (11.7)                                                                          2 (3.9)                                                                            0 (0.0)                                                                              1 (1.9)                                   MET 3 (5.0)                                                                            0 (0.0)                                                                            0 (0.0)                                                                            1 (1.3)                                                                            0 (0.0)                                                                            0 (0.0)                                                                              0 (0.0)                                   PHI 4 (6.7)                                                                            7 (7.3)                                                                            3 (4.8)                                                                            3 (3.9)                                                                            1 (2.0)                                                                            1 (5.6)                                                                              1 (1.9)                                   PRO 3 (5.0)                                                                            4 (4.2)                                                                            3 (4.8)                                                                            4 (5.2)                                                                             6 (11.8)                                                                           5 (27.8)                                                                            4 (7.4)                                   SER 4 (6.7)                                                                            8 (8.3)                                                                            3 (4.8)                                                                            1 (1.3)                                                                            3 (5.9)                                                                             2 (11.1)                                                                            5 (9.3)                                   THR 2 (3.3)                                                                            4 (4.2)                                                                            5 (8.1)                                                                            6 (7.8)                                                                            1 (2.0)                                                                            0 (0.0)                                                                               8 (14.8)                                 TRP 0 (0.0)                                                                            1 (1.0)                                                                            1 (1.6)                                                                            2 (2.6)                                                                            4 (7.8)                                                                            0 (0.0)                                                                              1 (1.9)                                   TYR 2 (3.3)                                                                            0 (0.0)                                                                            3 (4.8)                                                                            7 (9.1)                                                                            1 (2.0)                                                                            0 (0.0)                                                                              4 (7.4)                                   VAL 4 (6.7)                                                                            7 (7.3)                                                                            2 (3.2)                                                                            3 (3.9)                                                                            2 (3.9)                                                                            1 (5.6)                                                                              0 (0.0)                                   __________________________________________________________________________

Although the E18 cDNA 48-1 is incomplete, we found that exons 1-6 (FIG.9) hybridize to the 1.8 kb mRNA as well as to the 1.6 kb mRNA onNorthern blots. The structure of the two RNAs is most likely identicalup to exon 7. The additional splicing from the middle of exon 7 to exon8 characterizing the E18 cDNA, was confirmed by sequencing theseintron-exon boundaries in the genomic DNA clone (Table 2). The truncatedexon 7a present in the E18 cDNA is followed by a 1.6 kb introncontaining the polyadenylation site of the 1.6 kb RNA. Exon 8 begins 98bp downstream from the unique BamHl site of the gene (Table 2, FIG. 9).The genomic exon 8 ends by the polyadenylation site of the 1.8 kb RNA,characterized by a tandem repeat of the AATAAA signal. Athough exon 7and 8 have no homology, a conserved undecanucleotide ACCATTTATTG, inwhich the third cytidine is polyadenylated, is present at the end ofboth exons (Table 2). As pointed out previously (Benech et al., 1985), ahairpin-loop structure can be formed in both cases between thisconserved and undecanucelotide and the AATAAA region; such structuresmay participate in the cell-specific mechanism which determines whethercleavage and polyadenylation of the transcripts occur at the end of exon7 or at the end of exon 8.

Based on the above gene mapping, the enzyme coded for by the 1.8 kb mRNAshould be identical to the E16 product in the first 346 amino acids,which are followed by a specific 54 amino acid-long region, rich inaspartic acid, glutamic acid and threonine. The 400 amino acid-long E-18enzyme would have a molecular weight of 46,000.

EXAMPLE 7 Two forms of the human (2'-5') oligo A synthetase produced byalternative splicing of the same gene

Northern blot analysis of SV80 RNAs revealed that 3 species of RNA (1.6,1.8 and 3.6 kb) hybridizing to El cDNA accumulate in cells up to 12hours after exposure to IFN (Merlin et al., 1983). Additional unstabletranscripts were also seen. The relationship between these RNAs wasinvestigated by transcript mapping on genomic DNA clones. In two humangenomic libraries, the El cDNA identified only one series of overlappinggenomic DNA clones which represent 29 kb of human DNA (FIG. 9A) and werefound to contain an apparently unique (2'-5') oligo A synthetase gene(Benech et al., 1985a). By Sl nuclease analysis and partial genesequencing, the 9-21 (El) cDNA was found to correspond to 5 exons(numbered 3-7 on FIG. 9A and in the sequence of FIG. 7). The 3' end andpolyadenylation site of this cDNA was identified at the end of exon 7(FIG. 9). However, hybridization of further downstream genomic DNAfragments to Northern blots of SV80 RNA, revealed (Benech et al., 1985)that only the 1.6 kb RNA ended at the polyadenylation site in exon 7,while both the 1.8 and 3 6 kb RNAs hybridized to an additional exonlocated 1.6 kb downstream and which ends also by a polyadenylation site(exon 8, FIG. 9). Thus the 9-21 (El) cDNA represents the 1.6 kb RNA andwas renamed E16 cDNA. It was further found that the 3' half of exon 7does not hybridize to the 1.8 kb RNA indicating that the transcript isformed by a splicing event from the middle of exon 7 to exon 8. All the5' upstream exons hybridized to both 1.6 and 1.8 kb RNAs, indicatingthat the 2 RNAs differ only in their 3' ends. This was confirmed by theisolation from the SV80 lambda-gt10 cDNA library of a cDNA clone for the1.8 kb RNA (clone 48-1 or E18 cDNA, FIG. 7B), which demonstrated thedifferential splicing and ended at the polyadenylation site of exon 8(FIG. 9). A similar cDNA clone was found in a Daudi cDNA library bySaunders and Williams (1984). The E18 sequence locks the last 247nucleotides of E16 which are replaced by 515 nucleotides accounting forthe difference in size between the 1.6 and 1.8 kb RNAs.

The 1.8 kb RNA would thus code for a 46,000 Mr protein (E18) whichdiffers from the E16 protein in its C-terminus. Like the E16 protein,the E18 product has ds RNA binding and (2'-5') oligo A synthetaseactivity as shown by translation of mRNA selected by hybridization toE18-specific DNA fragments (Benech et al., 1985). This suggests that thefirst 346 aminoacids common to the 2 proteins contain the catalyticsites. Examining the exon composition this common part appears composedof a N-terminal acidic domain, followed by three basic regions. The last18 residues of the E16 protein form a very hydrophobic domain, which isreplaced in E18 by a longer hydrophilic and acidic region which alsocontains a potential glycosylation site. This difference between the 2enzymes may determine their ability to dimerize, or interact with otherproteins and cellular structures. For example, E16 may bind to membraneswhile E18 may interact with basic proteins in ribosomes or in thenucleus.

Two forms of the (2'-5') oligo A synthetase were found by gel filtrationin extracts of IFN-treated human cells (Revel et al., 1982): a 30-40 kdenzyme which could correspond to a monomeric form of the E16 or E18proteins, and a 60-80 kd enzyme which remains to be identified. The 3.6kb RNA does not seem to code for a large enzyme since transcript mappingshowed that this RNA contains intronic regions (e.g. between exon 7 andwhich are removed from the 1.8 kb RNA and have no open reading frame Wealso failed to see large E mRNA in oocyte translations. An 80 kd proteinin SDS was reported in purified human (HeLa) synthetase (Yand et al.,1981) but is enzymatic activity was not demonstrated. In enzyme purifiedfrom Namalava and CML cells (Revel et al , 1981b) we could detect a 40kd band in SDS. Thus it remains possible that the 60-80 kd enzyme formis a dimer of the 40 kd protein. The human synthetase may differ fromthat in mouse cells where a large 3.8 kb RNA was seen under denaturingconditions which codes for a 80 kd enzyme (mainly cytoplasmic), inaddition to a 1.5 kb RNA coding for a 30 kd enzyme (mainly nuclear) (St.Laurent et al., 1983). The human E cDNA detects a 3.8-4 kb and a 1.6-1.7kb RNA in mouse cells, the large RNA hybridizing to E18-specific DNA(Mallucci et al., 1985). It is possible that in human cells the largeRNA is further processed into 1.8 kb RNA, which has not been seen inmouse cells. Shulman et al. (1984) have used the fact that the bulk ofthe (2'-5') oligo A synthetase in human cells behaves as a smallerprotein than in mouse cells to map the human synthetase gene tochromosome 11 in human rodent-hybrid cells. Antisera specific to E16 andE18 will help to elucidate the relationship between these proteins andthe two forms of the native enzyme seen in human cells.

EXAMPLE 8 Cell specific expression of the two (2'-5') oligo A synthetasemRNAs

RNA from a number of human cell lines have been examined in Northernblots with the E cDNA probe (Merlin et al., 1983; Benech et al., 1985).Table 4 shows that human cells can be grouped in 3 classes according tothe predominant E mRNA species induced by IFN. Lymphoblastoid B celllines from Burkitt lymphomas have mainly the 1.8 kb RNA. Instead,several cell ines have the 1.6 and 3.6 kb RNA but little 1.8 kb RNA. Ifthe 3.6 kb RNA is a partially spliced precursor of the 1.8 kb RNA, thesecells may have an inhibition in the processing of the 3.6 kb RNA.T-lymphocyte lines (CEMT from an acute leukemia and Gash from hairy cellleukemia) contain like fibroplastic cells, all 3 E RNA species. The E18polyadenylation (pA) site seems, therefore, to be used in all humancells to produce either 3.6 or 1.8 kb RNA. The E16 pA site seems not tobe used in B lymphoblastoid cells. A conserved undecanucleotide presentin both E16 and E18 pA sites (FIG. 9B) can form a hairpin-loop with theAATAAA signal and could have a role in site selection (Benech et al.,1985). E18 has a tandem repeat of the AATAAA signal (FIG. 9B) and couldbe a stronger pA site. Transcripts ending at the E18 pA site accumulateearlier after IFN addition than the 1.6 kb RNA (Benech et al., 1985).

                  TABLE 4                                                         ______________________________________                                        PREDOMINANT (2'-5') OLIGO A SYNTHETASE                                        RNA SPECIES                                                                   ______________________________________                                         3.6 kb                      3.6 kb                                                         1.8 kb         1.8 kb                                            1.6 kb                      1.6 kb                                                        B lymphoblastoid                                                 Histiocytic  Burkitt lymphoma:                                                                            Fibroblastic:                                     lymphoma U937                                                                 Daudi                                                                         SV 80                                                                         Namalva                                                                       FS11                                                                          Amniotic Wish                                                                 Raji                                                                          Cervix Ca HeLa              T cells:                                          Raji × HeLa hybrids                                                     CEMT                                                                                                      Hairy cell-leuk.:                                 Gash                                                                          ______________________________________                                    

The type of synthetase predominantly made may vary in different humancells. We found no correlation between the cytoplasmic or nuclearlocalization of the synthetase and the type of RNA present in the cells.However, Namalava cells seemed to have mainly the 30-40 kd enzyme upongel filtration while HeLa and SV80 cells had also the 60-80 kd form(Revel et al., 1982).

EXAMPLE 9 Promoter region of the (2'-5') oligo A synthetase gene

The Sphl-Sohl fragment of 0.85 kb (FIG. 10) from the genomic 4.2 kbEcoRl fragment (FIG. 9) which contains part of exon 3 of the E16 cDNA9-21 clone, hybridized in Northern blots with the 1.6, 1.8, 2.7 and 3.6kb RNAs. However, upstream regions did not. Several experiments allowedto localize the RNA transcriptional start in this fragment. Sl nucleaseanalysis first showed that exon 3 starts about 50 nucleotides upstreamof the end of the 9-21 cDNA. A primer extension experiment using anoligonucleotide from the end of the 9-21 cDNA, indicated that the 5' endof the mRNA is about 230 nucleotides from the 5' end of this cDNA. RNAhybridization with riboprobes produced in SP6 (Green et al., 1983) andRNAse digestion indicated two exons of 70 and 110 nucleotides precedingexon 3. By Sl nuclease analysis using a probe labeled at the unique Hpalsite (FIG. 9), the 5' end of the mRNA was finally located 17 nucleotidesupstream from the Hpal site. The sequence of this region is shown inFIG. 11. The location of the transcription initiation site 17 residuesbefore the Hpal site, is supported by the presence of a TATAA box atposition -30. A striking feature of the upstream sequences, is the highpurine content (69.6%) mostly adenine (58.9%). Run of a homology matrixwith other known promoter upstream sequences revealed a surprisinghomology with the human IFN promoters in particular with the sequence ofthe IFN-beta-1 gene promoter (Degrave et al., 1981). The purine-richregion from -75 to -85 of the IFN-beta-1 promoter, which contains theessential transcription signal described by Zinn et al., (1983), shows90% homology with the region of the presumed promoter of the (2'-5')oligo A synthetase just upstream of the TATAA box (-40 to -50) (FIG.11). This purine-rich signal is repeated in the IFN-beta-1 promoter inthe segment between the TATAA box and the cap site; in this region,which may also have regulatory functions (Nir et al., 1984) the homologybetween the IFN-beta-1 gene and the (2'-5') oligo A synthetase gene ishigh. In contrast, search for homology with promoters of other genes,such as HLA genes (Malissen et al., 1982; Schamboeck et al., 1983) andthe metallothionein II gene (Karin and Richards, 1982) which areactivated by IFNs (Fellous et al., 1982; Rosa et al., 1983b; Friedman etal., 1984) showed no apparent sequence relationship in this region ofthe (2'-5') oligo A synthetase gene promoter. Also, no significanthomology was seen with the body of the IFN-beta-1 gene.

The 5' untranslated leader of the (2'-5') oligo A synthetase mRNA (exon1, 2 and part of exon 3) contains two short introns whose positions weretentatively determined by Sl analysis as shown in FIG. 11. The entirehuman (2'-5') oligo A synthetase gene is about 13 kb (FIG. 9) and thesum of the exons agrees with the observed sizes of the mRNAs.

EXAMPLE 10

Lambda GT10 cDNA clones of the (2'-5') oligo A synthetase

A lambda-gt10 cDNA library prepared from poly A+ RNA of human SV80 cells(Wolf and Rotter, 1985) was screened using as probe the Pstl-Pstl insertof the El cDNA plasmid described previously (Merlin et al., 1983). Theinsert corresponding to the 3' end of the 1.6 kb E RNA (Benech et al.,1985), was purified by agarose gel electrophoresis and nick-translated(Rigby et al., 1977). Plaques were repeatedly picked from 9cm plates(10⁵ phages), and small scale lambda-DNA preparations were analyzed byrestriction mapping by routine procedures (Maniatis et al., 1982).Fifteen lambda-gt10 cDNA clones containing the El cDNA fragment wereisolated and phages 9-2 and 5-2 with the longest inserts were cut byEcoRl and the inserts subcloned into pBR322 to obtain E16 cDNA clones9-21 and 5-21 of FIG. 6A. The same library was rescreened with a humangenomic Pstl-Pstl 0.9 kb fragment from phage lambda-chEl (Benech et al.,1985), a fragment which specifically hybridizes to the 1.8 kb RNA. Wethereby isolated lambda-gt10 cDNA clone 48-1 of FIG. 6B, along withanother cDNA clone representing a partially spliced E RNA. Sequencingwas carried out according to Maxam and Gilbert (1980). Restrictionenzymes were from New England Biolabs and Boehringer. Homology matrixand hydropathy plot computer programs of Pustell and Kafatos (1982a,b)were run on an IBM PC. Three base periodicity to locate protein codingframes was computed according to Trifonov (1984).

EXAMPLE 11 Genomic DNA clones containing the (2'-5') oligo A synthetasegene

Three overlapping genomic clones were isolated as previously described(Benech et al., 1985): lambda-chEl from a partial EcoRl-cut DNA library(Mory et al., 1981) and lambda-chE2 and E3 from a partial Alul/Hae 3 DNAlibrary (Maniatis et al., 1978). The genomic EcoRl fragments of thesephages were subcloned in pBR322. Exon mapping was done: 1) by Southernblot hybridization of restriction digests from subcloned genomicfragments to various cDNA probes; 2) by hybridization of genomic DNArestriction fragments to Northern blots of poly A+ RNA from IFN-treatedand untreated cells as described (Benech et al., 1985); and 3) bysequencing of intron-exon boundaries in comparison to cDNA. The internalSohl-Sphl 0.87 kb segment of the genomic 4.2 kb EcoRl fragmentcontaining the 5' end of the mRNA, was subcloned in the Sohl site ofpBR322 before sequencing. Primer extensions using syntheticoligodeoxyribonucleotides of 18-20 bases complementary to the mRNA (giftof Dr. D. Segev, InterYeda) were done as before (Rosa et al., 1983 a).Riboprobe synthesis after subcloning in the SP6 vector was carried outaccording to instructions of Promega Biotec. DNA from Daudilymphoblastoid cells and from FSll foreskin fibroblasts was preparedaccording to Wigler et al. (1979) and Southern blot analysis was done onGene-Screen Plus nylon fiber sheets using hybridization procedure Brecommended by the manufacturer (New England Nuclear).

EXAMPLE 12 Quick cell blot assay of (2'-5') oligo A synthetase RNAs forthe clinical monitoring of IFN action

The usefulness of measuring the (2'-5') oligo A synthetase has beenshown in human peripheral blood mononuclear cells (PBMC) to monitor theresponse of patients to IFN-beta (Schattner et al., 1981a) and IFN-betai.m. injections (Schoenfeld et al., 1984). Since the enzyme level ofPBMC in normal healthy individuals is rather constant, this assay hasallowed the diagnosis of viral infections evidenced by an increase inthe enzyme in the PBMC and granulocytes (Schattner et al., 1981b, 1984;Schoenfeld et al., 1985). Decrease in the enzyme characterize acuteleukemias with numerous blast cells (Wallach et al., 1982; Schattner etal., 1982). This technique has also been pioneered by Williams et al.(1981) and is now in wide use.

Synthetase E is strongly induced in cells treated by all three types ofIFNs, alpha, beta and gamma, and its increase is a good marker of IFNactivity (Wallach et al., 1982). It is therefore possible to usemeasurements of E levels to determine whether cells in vitro or in vivohave been exposed to IFN and respond to it. This measurement may be usedas an assay for IFN in unknown solutions, by exposing cells to saidsolutions and determining the increase in E levels (Revel et al., U.S.Pat. No. 4,302,533). The measurement may also be used to establishwhether IFN is produced in increased amounts in whole organismsincluding man.

The (2'-5') oligo A synthetase increases during differentiation ofhematopoietic cells as a result of autocrine secretion of IFN-beta(Yarden et al., 1984). Another important application of E measurementsis in the monitoring of patients under IFN therapy. Besides clinicalchanges, it is possible to establish that the patients respond to IFN bymeasuring the PBMC E level which increases 5-10 fold during systemicIFN-alpha as well as beta treatment (Schattner et al., 1981a; Schoenfeldet al., 1984). It is clear that assay of other IFN-induced activities ormolecules may be used as well as the assay of the E enzyme, but thismethod has been the most widely used (Williams et al., Borden).

Now the assay of E RNA in human PBMC is used for the same purpose. Aquick cell blot (Cheley and Anderson, 1984) using the 9-21 E cDNA asprobe was developed for PBMC (FIG. 14). Oligonucleotides derived fromthe E cDNA may also be used as probes. The effect of 10 U/ml IFN caneasily be detected by this method (FIG. 15). Positive signals wereobtained in a patient treated by 10⁷ units/day of IFN-alpha-c.

EXAMPLE 13 Immunoprecipitation of the (2'-5') oligo A synthetaseactivity by anti-(2'-5') oligo A synthetase peptide antibodies

Antiserum B was raised against a peptide common to the E16 and the E18sequences, while antiserum C was raised against a peptide found only inE16. We used these antibodies to verify that they immunopreciptate the(2'-5') oligo A synthetase activity specifically. The (2'-5') oligo Asynthetase activity adsorbed on the immune IgG-Protein A Sepharose andthat remaining in the supernatant were compared to the same fractionsobtained by using non-immune IgG. Extracts from two cell lines whichexpress preferentially either the 1.6 kb RNA (Wish cells) or the 1.8 kbRNA (Daudi cells) (Benech, et al., 1985a) were compared. Antibodies C(E16-specific) were 20 times more efficient to adsorb the activity fromWish cells than normal serum. Substracting the background with normalserum, allows to evaluate what is specifically bound to anti-B andanti-C (FIG. 16). Anti-C retained the (2'-5') oligo A synthetaseactivity from Wish cells but not from Daudi cells, in the line with theabsence of E16 mRNA and 40 kd protein in these cells (see Example 14).Anti-B adsorbed (2'-5') oligo A synthetase activity from both Daudi andWish cell extracts.

The antibodies produced against peptides deducted from the cloned cDNAs,recognized, therefore, specifically different (2'-5') oligo A synthetaseforms.

EXAMPLE 14 A) Immunoblot analysis of the different forms of (2'-5')oligo A synthetase from human cells

The antibodies against peptide B were tested for their ability to bindspecifically to (2'-5') oligo A synthetase in crude extracts of humancells separated by sodium-dodecyl-sulphate-polyacrylamide gelelectrophoresis and blotted electrophoretically onto nitrocellulosepaper. In additional to being recognized by the antibodies inimmunoblots, we expect genuine (2'-5') oligo A synthetase proteinspresent in extracts of these human cells to be induced by IFN treatment,and we therefore looked only at the induced proteins revealed by theimmunoblots (FIG. 17).

The cell line Daudi, Wish and SV80 were compared because of theirdifferences in the pattern of expression of the (2'-5') oligo Asynthetase mRNAs (see Example 13). Antibodies B detect as expected a45-46 kd protein similar in size to the E18 product in Daudi cells andno 40 kd which would correspond to E16 whose mRNA is not expressed byDaudi cells. In contrast, the 40 kd E16 protein is present in Wish cellswithout 46 kd E18 in line with the absence of 1.8 kb RNA in these cells.Both proteins are detected by anti-B in SV80 cells. These resultsdemonstrate that human cells produce the 40 and 46 kd proteins and thisonly when they express the 1.6 and 1.8 kb RNAs respectively.

The immunoblots with anti-B also reveal that there are not only twoforms of (2'-5') oligo A synthetase in human cells but probably fourdifferent forms. This can be deduced from the fact that in addition tothe 40 and 46 kd proteins, anti-B clearly detected two other proteins of100 kd and 67 kd which are induced by IFN (FIGS. 17 and 18). The 100 kdwas not detected in Daudi cells, showing that the large proteinsdetected by anti-B are also expressed in a cell-specific pattern. Thefact that the anti-B was raised against a peptide derived from thesequence of geniune (2'-5') oligo A synthetase forms, and that the twolarger proteins are induced by IFN, makes it very likely that theybelong to the (2'-5') oligo A synthetase system. To ascertain that theseare (2'-5') oligo A synthetase forms, we have purified (2'-5') oligo Asynthetase activity from different cellular fractions of SV80 andfollowed in parallel the protein detected by antibodies B.

B) Separation of the different active forms of (2'-5') oligo Asynthetase

The separation of the different protein species detected by antibodies Bis shown in FIGS. 18 and 19. Most of the 40 kd protein remains in the100,00g supernant (S100) of NP40 cytoplasmic extracts from SV80 cells.It is not adsorbed on DEAE-cellulose in low salt and adsorbs toCM-cellulose from which it elutes at high salt concentration. Incontrast, the 100 kd protein is almost absent from S100 and isconcentrated in the microsomal pellet (P100) from which it can besolubilized by sodium deoxycholate (DOC) or 0.5M KCl. This protein wasadsorbed on DEAE-cellulose at low salt and elutes at high salt. The 67kd and 45-46 kd remain partly in S100 but are relatively concentratedper mg protein in the microsomal pellet. They appear to be less readilyextractable from microsomes by DOC or KCl. Sedimentation on glycerol9radients showed that the activity purified from S100 after CM-celluloseparallels the sedimentation of the 40 kd protein E16 The fractionpurified from P100 and eluted from DEAE-cellulose, containing the I00and 46 kd proteins, separated into two peaks on glycerol gradients,sedimenting as 80,000 and 45,000 Mr proteins The (2'-5') oligo Asynthetase activity in the heavy peak parallels the presence of the 100kd protein

C) Different enzymatic properties of the various (2'-5') oligo Asynthetase form

The 40 kd proteins from the glycerol gradient has an optimal pH of 6.8for its activity, and is only 25% as active at pH 7.8. Moreover, noactivity of the 40 kd (2'-5') oligo A synthetase can be observed atconcentrations of poly (rI)(rC) lower than 1μg/ml and the maximalactivity required 500-100μg/ml (FIG. 20). The same high ds RNArequirement was found for the E16 cDNA product produced by recombinantDNA technology in E. coli (not shown).

The 100 kd protein after the glycerol gradient, has an optimal pH of 7.6for its (2'-5') oligo A synthetase activity and is less active at acidicpH. It is maximally active already at extremely low concentrations ofpoly (rI)(rC) or in its absence, and its activity is even inhibited byhigh ds RNA concentrations This strongly suggests that the different(2'-5') oligo A synthetase forms, because of their different cytoplasmiclocalizations and enzymatic properties, are used by the cells underdifferent conditions.

Many observations suggest that the IFN-induced (2'-5') oligo Asynthetase involved in two distinct, seemingly opposite, phase of cellgrowth (cell-cycling and growth inhibition) in addition to its possiblerole in the antiviral effect (reviewed in Revel, 1984). This may berelevant to the issue of multiple (2'-5') oligo A synthetase forms. Insynchronized cell cultures we have observed that (2'-5') oligo Asynthetase behaves as a cell-cycle protein (Mallucci, et al., 1985).Thus, synchronized cultures of Mouse embryo fibroblasts exhibit a sharprise in (2'-5') oligo A synthetase activity and (2'-5') oligo Asynthetase mRNA at the end of the S-phase followed by a rapiddisappearance of the RNA and enzyme activity when the cells proceed toG2. Anti-mouse IFN antibodies reduced the (2'-5') oligo A synthetaseinduction. In this system we also observed that the (2'-5') oligo Asynthetase RNA which accumulates in S-phase is a large 4-5 kb transcriptdifferent from the 1.7 kb RNA species which accumulated in the samecells when treated with exogenous IFN. This suggests that the S-phase(2'-5') oligo A synthetase is a different form of the enzyme than thatin cells growth-arrested by exogenously added IFN. Because of its largemRNA it is likely to be like the 100 kd, a low ds RNA requiring form.Anti-B antibodies also detected the (2'-5') oligo A synthetase multipleforms in mouse cells.

These considerations illustrate the advantage of being able to assayindependently the 4 forms of (2'-5') oligo A synthetase, which may varyindividually in various physiological conditions and diseases

EXAMPLE 15 Use of anti-(2'-5') oligo A synthetase peptides antibodiesfor immunoassays of (2'-5') oligo A synthetase

Since the anti-B antibodies recognizes all the forms of (2'-5') oligo Asynthetase, it can be used for an immunoassay of (2'-5') oligo Asynthetase in unfractionated extracts of human cells either fromcultures or directly obtained from patients.

An example of a solid-phase radio-immunoassay is shown in FIG. 21. Wishand Daudi cells, either treated by IFN or untreated, were lysed by theNP40 containing Buffer A and S15 prepared by microfuge centrifugation asdescribed above. Aliquots containing 1 to 10μg of protein were directlyapplied to nitrocellulose paper (or to other protein-binding paper) andthe sheet treated with anti-B as for regular immunoblots (Example 14).The autoradiography in FIG. 21 shows that ¹²⁵ I-Protein A binds only tothe samples originating from IFN-treated cells. It is clear that thisassay may also be used in the form of an enzyme-linked immunoassay(ELISA) by replacing the labeled Protein-A with peroxydase orβ-galactosidase conjugated antirabbit IgG.

The immunoassay of (2'-5') oligo A synthetase is rapid: 20 minutes forcell extract preparation; 2 hours for anti-B and Protein-A adsorptionand washing. The assay is sensitive and very small amounts of cellextracts suffice to measure the (2'-5') oligo A synthetase level. It isspecific, no signal being obtained in non-IFN treated cells.

EXAMPLE 1 Immunofluorescence microscopy detection of (2'-5') oligo Asynthetase in cells

Enzymatic assays have established that (2'-5') oligo A synthetase iselevated in peripheral blood mononuclear cells of patients with viralinfections. The anti-(2'-5') oligo A synthetase peptide antibodies ofthe present invention can be used in immunofluorescence microscopy todetect (2'-5') oligo A synthetase elevation in cells in general, andwhite blood cells in particular.

Blood (2 ml) was withdrawn from a healthy donor and from a patient withacute viral illness. The mononuclear blood cells were separated byFicoll-Hypaque (Pharmacia) centrifugation, and spread on glasscoverslips directly or by the use of a cytospin microfuge(microhematocrite). The cells were washed in PBS, fixed for 30 minutesin 3% paraformaldehyde at room temperature (RT), rinsed with PBS andtreated with 0.5% Triton-X 100 in Hank's salts for 5 minutes, rinsedagain with PBS and with PBS-2% gelatin. The coverslips were thenincubated with anti-B serum diluted 1:5 in PBS-gelatin applied as a 40μldroplet on parafilm onto which the coverslips were deposited. After 60minutes at RT, the coverslips were rinsed in PBS-gelatin and FITCconjugated anti-rabbit IgG (BioYeda) diluted 1:20 was applied by theparafilm procedure. After 20 minutes at room temperature, coverslipswere washed twice with PBS-gelatin, then with H₂ O and mounted onmicroscopic slides with Moviol 4-88 (Hoechst)-Glycerol (2.5g Moviol, 6gGlycerol and 6 ml H₂ O to which 12 ml of 0.2M Tris-HCl, pH 8.5, wereadded, followed by incubation at 50° C. and clarification 15 minutes at5,000Y). Parallel coverslips were processed using normal rabbit seruminstead of anti-B Slides were observed in a Zeiss fluorescencemicroscope and photographed on Polaroid film with 30 seconds exposures.

FIG. 22 shows that the lymphocytes were stained with anti-(2'-5') oligoA synthetase in blood samples from the patient with viral infection butnot in the blood of the healthy donor, where only a low fluoresence ofmacrophages and granulocytes is seen. Thus, the present anti-(2'-5')oligo A synthetase peptide antibodies can be used for microscopicobservation of cells, in cultures, blood and tissue sections, toevaluate if the cells have reacted with interferon and have accumulated(2'-5') oligo A synthetase.

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What is claimed is:
 1. An antibody against all four of the 40 kd, 46 kd,67 kd, and 100 kd forms of human (2'-5') oligo A synthetase obtained byimmunizing an animal with an antigenic peptide having an amino acidsequence consisting ofGLU-LYS-TYR-LEU-ARG-ARG-GLN-LEU-THR-LYS-PRO-ARG-PRO-VAL-ILE-LEU-ASP-PRO-ALA-ASP.2. A antibody of claim 11 conjugated with a label to formed a labeledantibody.
 3. A labeled antibody of claim 2, wherein the label is afluorescent label.
 4. A labeled antibody of claim 2, wherein the labelis a radioactive label.
 5. A labeled antibody of claim 2, wherein thelabel is an enzyme.
 6. A kit for the detection of all four forms ofhuman (2'-5') oligo A synthetase in cells comprising the antibody ofclaim
 2. 7. An antibody which specifically recognizes and binds to human(2'-5') oligo A synthetase and an antigenic peptide having an amino acidsequence contained within the amino acid sequence set forth in FIG. 7Acomprising consisting ofARG-PRO-PRO-ALA-SER-SER-LEU-PRO-PHE-ILE-PRO-ALA-PRO-LEU-HIS-GLU-ALA. 8.An antibody which specifically recognizes and binds to human (2'-5')oligo A synthetase and an antigenic peptide having an amino acidsequence contained within the sequence of amino acid 1-364 set forth inFIG. 7A consisting ofGLU-LYS-TYR-LEU-ARG-ARG-GLN-LEU-THR-LYS-PRO-ARG-PRO-VAL-ILE-LEU-ASP-PRO-ALA-ASP.